Collaborative trial validation of a new multiplex real-time PCR to sensitively detect allergenic nuts in food

In this article, we present a multiplex real-time PCR method for a simultaneous, sensitive and specific detection and semi-quantitative estimation of the allergenic species peanut, hazelnut, walnut and cashew in food. Due to the use of multicopy target sequences, a very sensitive detection of the allergenic ingredients was possible. The method was validated in-house as well as by a collaborative trial with 12 laboratories. Within the ring trial, 0.64 mg/kg (i.e. approx. 0.1–0.2 mg of peanut and tree nut-derived protein/kg) could still be detected in a processed cookie matrix, confirmed by results of incurred, processed samples spiked at very low levels between 0.9 and 50 mg/kg of the corresponding allergenic ingredient (peanut, tree nut). In addition, the method revealed good precision data. With regard to quantitative analysis though, insufficient recovery data (bias) were determined in some cases, resulting in measurement uncertainties of more than 50%.